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. 2019 May 8;15(5):e1008149. doi: 10.1371/journal.pgen.1008149

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Fig 5 — (A, B) Relative expression of ascorbate (AsA) biosynthesis, recycling and oxidation-related genes in fruits of SlbHLH59 overexpression (A) and RNAi (B) lines. (C) The promoter cis-elements of SlPMM, SlGMP2 and SlGMP3. The sequences in black boxes represent E-box cis-elements. (D) Yeast-one hybrid (Y1H) assay of SlbHLH59 binding to promoter fragments of SlPMM, SlGMP2 and SlGMP3. The bait vectors SlPMM_pro, SlGMP2_pro and SlGMP3_pro (the fragments containing the region 3000 bpupstream from initiation codon) and the prey vector containing SlbHLH59 were introduced into yeast strain Y187, and interaction between bait and prey enhanced ABA resistance. Yeast cells spread on SD-Leu media with various concentrations of ABA (0, 10, 15, and 20 mM). The bait vector (SlPMM_pro, SlGMP2_pro and SlGMP3_pro) + pGADT7 were also transformed into Y187 as a negative control. (E) Binding of SlbHLH59 to promoters of SlPMM, SlGMP2 and SlGMP3 assayed by dual luciferase system. The SlbHLH59 ORF was cloned into the effector vector (pGreen II62-SK) and promoter fragments of SlPMM, SlGMP2 and SlGMP3 were inserted into the reporter vector (pGreen II 0800 LUC). Both vectors of effectors and reporters were transformed into Agrobacterium cells and used to infiltrate tobacco leaves. LUC, firefly luciferase activity; RLU, Renilla luciferase activity; PG, the empty vector of pGreenII 62-SK. The promoters of SlPMM, SlGMP2 and SlGMP3 plus PG were used as control. (F-J) Dynamics of ascorbate content and SlbHLH59 expression during tomato fruit development. Ascorbate (AsA) concentration (F) and expression of SlbHLH59 (G), SlPMM (H), SlGMP2 (I), SlGMP3 (J) at different fruit developmental stages of wild-type (TS-186) and SlbHLH59 transgenic lines. Experiments were performed in immature (IMG), mature green (MG), breaker (BR), yellow ripe (YR) and red ripe (RR) fruit, respectively, with three replicates. All data in the graphs are presented as means ± SE. Asterisks indicate significant differences by t test: *P < 0.05; ** P< 0.01.